Chimeric antigen receptor (CAR)-engineered T cells are those T cells expressing chimeric receptors, which can recognize specific antigens and can transmit signals, expressed on the surface thereof [1]. CAR T cells play an important role in treating cancer by expressing CAR molecules, which typically include an extracellular domain, a transmembrane region and an intracellular domain: the extracellular domain is a single-chain variable fragment (ScFv) formed by a heavy chain and a light chain variable regions of an antibody that connected each other via a peptide fragment; the intracellular domain is comprised of a variety of signaling molecules, including CD3zeta, CD28, OX-40, 4-1BB etc.; and the transmembrane region is originated from the transmembrane region of other molecules (such as CD8, CD4, CD28 and CD3zeta). The single chain variable fragment gene is isolated from hybridomas capable of generating monoclonal antibody which targeting specific antigen. T cells expressing CAR molecules directly recognize tumor cell-surface antigens independent of the expression of the major histocompatibility antigen type I on tumor cells. Thus T cells expressing CARs can be activated by tumor specific antigen and kill tumor cells effectively. In short, CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition manner, and then experience activation, proliferation and exert cytotoxic function through their intracellular signaling.
Currently, clinical researches on CAR T cells mainly focused on hematologic tumor, such as lymphoma, chronic lymphoid leukemia, acute lymphoid leukemia. However, some progress has been made in developing CAR T cells for treating other types of tumors, for example, anti-5T4 CAR T cells for treating solid tumors, anti-ROR-1 CAR T cells for treating solid tumors, anti-PCSA CAR T cells for treating solid tumors, anti-Mesothelin CAR T cells for treating pancreas mesothelioma, anti-EGFRvIII CAR T cells for treating glioma and glioblastoma, anti-CD22 CAR T cells for treating B-cell tumor, anti L1CAM CAR T cells for treating neuroblastoma, anti-MUC16 & IL6 CAR T cells for treating ovarian cancer, etc. In summary, the immunotherapy of chimeric antigen receptor T cells has been proved to effectively treat a variety of tumors, including leukemia resistant to chemotherapy and relapsed leukemia. But there are many problems in chimeric antigen receptor T cell therapy, such as cytolytic activity, persistance of CAR T cell and the formation of memory CAR T cells in vivo/patients.
The structure of CAR molecules has developed for multi generations. The structure of the first-generation CAR molecules comprises a single chain variable fragment (scFv) recognizing the specific surface antigen of tumor cells, a transmembrane domain and a intracellular domain of CD3ζ (TCR complex) for activating T cells. As the intracellular domain of the first generation of CAR only has CD3ζ signal transfer region without co-stimulatory signals, there is a big flaw in the function of the first generation of CAR T cells, which exhibit low levels in proliferation, persistence, effector functions and so on in a patient. In order to enhance the function of the first generation of CAR in activating T cell, the second generation of CAR has been developed, and an intracellular molecular signaling domain originated from costimulatory molecules (e.g., CD28, CD134 (OX-40), CD137 (4-1BB), etc.) has been introduced to the intracellular domain of the second generation of CAR. Clinical trials show that the second generation of CAR T cells show good proliferation, persistence and effector functions in a patient. Most of the clinical trials of the second generation of CAR T cells are the treatment for B-cell leukemia with anti-CD19 CAR T cells. CAR T cells have achieved success for leukemia in clinical trials, but lack efficacy in solid tumors. In order to further improve the efficacy of the CAR T cell therapy, the third generation CARs have been developed. Two costimulatory molecule signal transfer regions were introduced into the intracellular domain of the third generation CARs. Typically, one costimulatory signal is the CD28 intracellular domain and the other is the intracellular signal transfer region of CD134, CD137 or ICOS, etc. Different combinations of the costimulatory signals may affect the function and efficacy of the CAR T cells, and studies have shown that not all of the third-generation CARs are better than the second generation CARs. It can be seen that the structural design of CARs in the prior art is not very mature, and there still exists a need for further improving CAR molecules in T cell expansion, T cell function of killing tumor cells, T cell invasion and migration, eliminating immunosuppressive effect of regulatory T cells and promoting the formation of memory T cells, etc.
Janeway, an American immunologist, proposed pattern recognition theory, in which the natural immune response against the main target molecules signal is called pathogen-associated molecular pattern (PAMP); the corresponding recognition receptor is called pattern recognition receptor (PRR). Toll-like receptors (TLR) are PRR which can specifically recognize molecules derived from different microorganisms with conserved structures, and activate intrinsic immune responses. Toll-like receptors are a class of important receptors involved in innate immunity, and also a bridge connecting adaptive immunity and innate immunity. Ten types of TLR have been found in human, and are called TLR1-10 respectively. Most of TLRs work alone, except that TLR2 can form dimers with TLR1 or TLR6 respectively to identify microbial exogenous molecules and host endogenous molecules. Increasing studies show that TLR signaling plays an important role in tumor development and treatment. Because some TLRs are widely expressed on the cell surface of immune cells, and different TLR plays different roles in these cells. The researchers find that TCR-activated human T cells express TLR2 but not TLR4, and TLR2 provides co-stimulatory signal for activating T-cell function and maintaining memory T cells [2]. The effects of TLR2 on CD8-positive T cells include: down-regulating TCR signal strength threshold required for activating T cells, prompting the formation of memory T cells under low TCR signal [7]. TLR1/TLR2 agonists can effectively eliminate the function of regulatory T cells by down-regulating the expression of Foxp3 [3, 4, 5]. Further studies show that, TLR1/TLR2 agonists can induce tumor regression by reducing the regulatory T cells and up-regulating the function of cytotoxic T cells [6].